Watermelon with improved processing qualities

ABSTRACT

A watermelon plant that produces fruit having (i) ultra-firm flesh and/or liquid-retaining flesh and (ii) soluble solids of at least about 6 brix.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/972,190 filed Oct. 22, 2004 now U.S. Pat. No. 9,173,356, which claimsthe benefit of provisional Application No. 60/584,964, filed on Jul. 2,2004, both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of the present invention is watermelon breeding and thegenetic improvement of watermelon. More specifically, this applicationis related to diploid, tetraploid and triploid watermelon seeds andplants for the production of watermelon fruit that (i) have ultra firmflesh and/or liquid-retaining flesh and (ii) are sweet at maturity.

2. Description of Related Art

Watermelon (Citrullus lanatus) is an important commercial member of theCucurbitaceae family that includes many different varieties. The fruitof these varieties differ in coloring, sweetness, and other traits. Forexample, watermelon fruit of different varieties display a wide range ofcoloring on the outside rind. In addition, color in the edible tissuevaries from different shades of red to yellow. Watermelon fruit alsovary in sweetness, which can be estimated by measuring total solublesolids, or brix, using a refractometer. Because sweetness is especiallyimportant to consumers, the U.S. Department of Agriculture has set fruitquality standards based on brix levels (United States Standards forGrades of Watermelon, U.S. Department of Agriculture (1978)). Accordingto these standards, edible parts of the fruit having not less than 8brix are deemed to be “Good”, while edible parts of the fruit having notless than 10 brix are deemed to be “Very Good.”

Consumers also have the choice of either seeded or seedless watermelonvarieties. Unlike the flesh coloring, which is caused by varying geneticloci, the distinction between seeded and seedless varieties is usuallycaused by human intervention of making crosses that vary ploidy levels.Similar to humans, watermelons are natural diploids with chromosomesarranged in pairs. Many plants, including watermelons, can undergo aduplication of their entire set of chromosomes and exist as tetraploids.While it is uncommon for watermelons to produce spontaneous tetraploids,this process can be routinely produced in the laboratory using cellbiology techniques. A tetraploid parent may then be crossed with adiploid parent to produce triploid seeds, which, in turn, generateplants with seedless fruits. In particular, seed formation in the fruitof triploid plants aborts because of the ploidy level differences,resulting in seedless fruits. Many commercial varieties are triploid andseedless.

Fruits of plants of different ploidy also vary in flesh firmness.Diploid lines typically have the lowest fruit flesh firmness levels. Forreasons that are unclear, the process of changing a diploid line to atetraploid line correlates with firmer fruit flesh. In other words,tetraploid lines usually have firmer fruit flesh than diploids.Triploids, being a cross between a tetraploid and a diploid, typicallyhave an intermediate level of flesh firmness.

In addition to consumer preferences as to coloring, sweetness and seeds,there is increasing consumer demand in the fresh produce business forproducts that combine quality and convenience. Examples of products thatmeet these criteria are bagged baby carrots, broccoli and cauliflowerand bagged leafy crops, such as lettuce and spinach. Similarly, there isdemand for mature cut fruits, like watermelon, melon, pineapple, papayaand kiwi. A growing segment of watermelon retail sales are cut fruitsthat are either displayed in large pieces with the rinds attached, orare cut into smaller pieces, without the rind, and offered to theconsumers in plastic food containers. The industry term for theseproducts is “minimally processed.” By 1998, Perkins-Veazie et al.((1998) Hortscience 33:605) estimated that 10% of the retail watermelonmarket was minimally processed.

The advantage of such cut fruit displays is that the consumer canvisually inspect the quality of the fruit and, in particular, judgewhether the fruit is mature and, thus, ready to consume. Often, immaturefruits will not be uniform in pigmentation, and overripe fruit willdisplay signs of decay. Moreover, these products offer convenience tothe consumer.

The disadvantage to the produce retailer in presenting minimallyprocessed watermelon products is that cut fruits have a short shelflife. Studies indicate that minimally processed products have a shelflife of 2 to 3 days maximum (ibidem; Wehner et al. In: Watermelons:Characteristics, Production and Marketing. Maynard, editor. ASHS Press,Alexandria Va. 2001.)

Watermelon fruits currently available typically undergo rapid qualitydeterioration after being cut. Cutting the fruit causes decay, which isobserved as a softening of the fruit texture. Deterioration is alsomanifested as liquid leakage; in some varieties, the flesh of a freshcut watermelon fruit quickly becomes unattractive to the consumer. Therapid deterioration of cut watermelon fruit places both time and spaceconstraints on the retailer. Because cut fruits have a short shelf life,the retailer typically performs the processing on the retail site. Inaddition, the retailer has to monitor the products often to ensure thatdeteriorating products are discarded.

Unlike the sweetness standards established by the U.S. Department ofAgriculture, there are no industry standards to describe the firmness ofthe edible portions of watermelon fruits. Therefore, there are a widerange of descriptors in use, from “firm” and “crisp” (Erma Zaden catalogdescriptors for varieties Gil 104 and Erma 12) to “very firm flesh”(Zhang et al. in USPTO application numbers 20040060085 and 20030217394and Seminis watermelon catalog for the variety Cooperstown). Seminis hasdescribed cultivars Fenway, Royal Star and Sentinel as having “excellentcrispness,” “firm flesh” and “crisp juicy flesh,” respectively. Inaddition, Rogers Seed Company advertises the Tri-X Brand 626 as“exceptionally firm” and the Tri-X Brand 313 as having “firm texture”and “crispness.”

While advertising terminology used to describe watermelon fruit fleshfirmness is quite variable, scientific reports, using quantitativemeasurements, show that typical commercial germplasm have hadsubstantially lower flesh firmness than the watermelon fruit of thisinvention. For example, Roberts et al. (2004 Report from: WatermelonResearch and Development Working Group. 24^(th) Annual Meeting, Tulsa,Okla.) measured flesh firmness in a wide range of germplasm, using apenetrometer to measure the amount of force resisted. The data werereported in Newtons, an International System of Measurements term. Forpurposes of comparison with Applicants' penetrometer measurements,Applicants converted Roberts' data to pounds force (lbf), using thefollowing formula: 1 lbf=4.448 Newtons. Roberts reports a range ofwatermelon flesh firmness between approximately 1.4 to 3.4 lbf. One ofthe lines analyzed is Rogers Seed Company line Tri-X Brand 313. As notedabove, Rogers Seed Company advertises this line as having “firm” flesh.Roberts et al. measured the flesh firmness in Tri-X Brand 313 as 10.84Newtons, which converts to approximately 2.4 lbf. Applicants also testedthe flesh firmness of Tri-X Brand 313, using a penetrometer from QASupplies in Norfolk, Va. (Model FT011) with a probe diameter of 8 mm.Using this methodology, Tri-X Brand 313 has a flesh firmness reading of1.4 lbf (Table 1). Because Roberts does not report the size of thepenetrometer probe used, Applicants cannot directly compare their datato Roberts'. At least for Tri-X Brand 313, the approximately 77% higherreading measured by Roberts et al. compared with the protocol describedherein may be the result of different methodology, and, in particular,the use of differently sized penetrometer probes. Although theApplicants of this invention use an 8 mm probe, another commonly usedpenetrometer has a diameter of 11 mm, which would account for thedifferent readings, as penetrometer area is approximately 73% higher foran 11 mm probe as compared to an 8 mm probe.

Schultheis and Thompson (2004 Report from: Watermelon Research andDevelopment Working Group. 24^(th) Annual Meeting, Tulsa, Okla.) alsosurvey watermelon fruit flesh firmness. Although these authors use adifferent model penetrometer than that used by Applicants, they use avery similarly sized probe with a diameter of 5/16″ or about 8 mm.Schultheis and Thompson report that line Tri-X 313 had flesh firmnessreadings between 1.4 and 1.7, which are similar to Applicants'measurements, shown in Table 1. In this report, however, the authorsdescribe these firmness data in units of pounds/square inch. It issuspected, however, that the units provided in the Schultheis andThompson report should be in pounds force, as a reading of 1.4pounds/square inch, using a 5/16″ probe, is only 0.15 pounds force.

Maynard and Sidoti (2003 GCREC Research Report BRA-2003; Univ. Florida,Gulf Coast Research and Education Center, Bradenton, Fla.) report anadditional survey of fruit flesh firmness of commercial watermelonlines. In this study, the authors use a different model penetrometerthan that Applicants use in the method described herein, with a largersized probe having a diameter of 7/16″ or about 11 mm. Their firmnessdata range from 1.8 to 3.0 pounds/square inch. As with the Schultheisand Thompson report, Applicants believe that these authors are using theincorrect units in their firmness readings. Assuming that these data areactually in pound force units, they compare well with the resultsobtained using the methodology described herein. For example, Maynardand Sidoti's firmness measurement of line Tri-X 313 was 2.6. If oneadjusts this figure to correct for the approximate 2 times difference inprobe area, the new figure is 1.35, which is nearly identical toApplicants' measurement of this same line, (Table 1). On the other hand,if one assumes that the data are correctly reported in lb/square inch,the figure of 2.6 lb/square inch based on a 7/16″ probe would be readingof 0.39 lbf. The Tri-X 313 line should have a much higher firmnessreading than 0.39 lbf, providing further evidence of inconsistency inhow such units have been reported in the prior art.

Leskovar et al. ((2004) J. Horticultural Science and Biotechnology 79:75-81) also report watermelon fruit firmness. Although this manuscriptuses a different measurement protocol, the authors describe in detailtheir methods, allowing the data to be converted for comparison with thedata described herein. After converting to the same units, the range ofgermplasm analyzed had fruit firmness between 0.9 lbf and 1.5 lbf.

Although measurements of the prior art can be confusing, there isclarity that commercial watermelon lines produced prior to thisinvention have fruit firmness that is well below 3 lbf. In addition, asshown in Example 4, the fruit of such commercial watermelon lines, oncecut, undergo significant liquid leakage. The present invention,therefore, addresses the need in the marketplace for watermelon linesthat produce fruits that have a longer shelf life when processed.Specifically, the watermelon of this invention have (i) ultra firmflesh, which avoids the problem of cut fruit becoming overly soft,and/or (ii) liquid-retaining flesh, which delays deterioration of cutfruit by liquid leakage. In addition, these fruits have qualitycharacteristics desired by the consumer, such as sweetness andattractiveness, and offer the retailer both flexibility as to wherefruit processing occurs and additional shelf life once fruit isprocessed.

SUMMARY OF THE INVENTION

This invention relates to unique watermelon inbred lines and hybridvarieties that produce fruit having ultra firm edible flesh at maturitythat resists at least 3.0 Pounds force (lbf) (measurement techniquesdefined herein). In addition to the novel ultra firm flesh phenotype,these fruits meet market requirements for sweetness, having not lessthan 6 brix for the edible tissue (measurement techniques definedherein).

Watermelons of this invention are preferably diploid and tetraploidinbred lines that produce sweet tasting ultra firm flesh at maturitythat resists at least 3.5 lbf, though lines that produce sweet tastingultra firm flesh at maturity that resists at least 4, 5, 6 and even 8lbf are also contemplated by this invention. A plurality of watermelonplants grown in a field are also provided by the invention.

Any diploid or tetraploid inbred line having ultra firm flesh createdfrom the teachings of this invention can transmit this ultra firm fleshphenotype to a hybrid. In addition to having ultra firm flesh atmaturity, the watermelons of the present invention are capable ofdeveloping uniformly pigmented fruit flesh (red, yellow, or orange). Inaddition, at maturity, fruits from these inbred lines and hybrids willmeet or exceed industry standards for sweetness, being at least good(not less than about 8 brix) and preferably very good (not less thanabout 10 brix).

The invention also provides a method for producing hybrid watermelonseed comprising crossing an inbred watermelon plant with a secondwatermelon plant and harvesting resultant hybrid watermelon seed, aswell as a hybrid watermelon plant produced by growing the resultanthybrid watermelon seed.

The invention further provides a method for producing the ultra firmwatermelon plant comprising the steps of crossing a watermelon varietyhaving a level of sweetness that at least meets industry standards witha low sweetness watermelon variety having ultra firm flesh; performingat least one backcross with the variety having a level of sweetness thatat least meet industry standards, and performing one or more cycles ofself-pollination of products of the backcross (or recurrent backcross)having the combined traits of ultra firm flesh and sweetness that atleast meets industry standards. The method may utilize as a watermelonhaving ultra firm flesh the watermelon plant of USDA Collection No.PI296341.

Watermelon fruit and watermelon flesh derived from the ultra-firmwatermelon are also contemplated. Preferred are watermelon plantsproducing a fruit weighing at least about 1.5 kg, more preferablyproducing a fruit weighing at least about 3.0 kg. In a further preferredembodiment the watermelon plant produces a fruit weighing at least about4.5 kg, and in a still further preferred embodiment the plant produces afruit weighing at least about 6.0 kg.

The invention also provides a watermelon plant having the soluble solidsand flesh firmness traits of a plant produced from seed deposited asAccession No. NCIMB 41230, made on Jul. 1, 2004, as well as seed,pollen, ovule and other vegetative tissue derived from the plant, or awatermelon plant regenerated from such tissue.

The invention also provides a watermelon plant with liquid-retainingflesh. As explained in detail below, this liquid-retaining traitcorresponds to the amount of weight that cut watermelon fruit fleshloses over time. Preferred are watermelon plants wherein cut flesh fromthe watermelon fruit loses less than about three and one-half percent ofits weight after three days storage at 4° centigrade. More preferred aresuch watermelon plants where the cut flesh loses less than about threepercent weight after three days storage at 4° centigrade. A stillfurther preferred watermelon plant is provided where the cut flesh losesless than about two percent weight after three days storage at 4°centigrade. In another preferred embodiment, the watermelon plant hascut flesh that loses less than about one and one-half percent weightafter three days storage at 4° centigrade. This liquid-retaining traitextends the shelf life of processed watermelon fruit.

A preferred embodiment is a good ultra firm flesh watermelon diploidinbred line that produces sweet tasting mature fruit. Another preferredembodiment is a triploid hybrid, created using as at least one parentalline that is either an ultra firm flesh diploid inbred line or an ultrafirm flesh tetraploid inbred line that produces good standard sweettasting mature fruit with ultra firm flesh. In another preferredembodiment the mature watermelon fruit produced in the diploid,tetraploid, or triploid plants of this invention develop full red fleshcolor and are sweet tasting, with good brix levels.

In yet another preferred embodiment, mature watermelon fruits of thisinvention develop full yellow flesh color and good sweetness incombination with ultra firm flesh. In still yet another preferredembodiment, the mature watermelon fruits of this invention develop fullorange color and good sweetness in combination with ultra firm flesh. Inanother preferred embodiment the watermelon flesh from fruits of thisinvention stays ultra firm after being minimally processed (fresh cutfruit). This ultra firm feature extends the shelf life of the processedfruit.

The present invention also relates to a novel method of producingdiploid and tetraploid watermelon lines and triploid watermelon hybridsthat produce sweet tasting mature fruit with ultra firm flesh (resistspressure of at least 4.0 lbf; not less than 8 brix).

One step in this method involves crossing a known watermelon variety orline with a watermelon line of this invention having ultra firm flesh atmaturity. The product of such cross is then self-pollinated to create asegregating population. In successive generations, individuals frompopulations segregating for the ultra firm flesh trait are subjected tosuccessive cycles of selection and breeding and the end result is a newwatermelon line that produces sweet tasting mature fruit having ultrafirm flesh.

Other objects, features and advantages of this invention will becomeapparent from the detailed description that follows. It should beunderstood that the detailed description and examples, while statingpreferred embodiments of the invention, are by way of illustration only,as modifications and changes within the scope of the invention willbecome apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a histogram that illustrates fruit flesh firmness of the thirdgeneration of self-pollinated inbred watermelon plants of the presentinvention. The arrow indicates the average mature fruit firmness of therecurrent parent lines. The shaded portion of the histogram shows that43% of these fruits have firmness readings at or above 4 lbf.

FIG. 2 is a graph showing weight loss after storage at 4° centigradeamong processed fruit of standard commercial watermelon varieties andprocessed fruit of watermelon of the present invention. The weight lossclosely approximates liquid leakage from the processed fruit.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a watermelon plant that produces fruitwith (i) ultra firm flesh and/or liquid-retaining flesh and (ii)sweetness of at least 6 brix. Therefore, the fruit of this inventionhave improved processing qualities, as, once cut, the fruit remains firmand/or retains its juice considerably longer than the commercialwatermelon lines of the prior art.

Definitions

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which,watermelon plantscan be regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants such as pollen, flowers, seed,leaves, stems and the like.

As used herein, “diploid plants” means plants or transplants derivedfrom planting diploid seeds or from micropropagation that have two setsof chromosomes in the somatic cells, or twice the haploid number.

“Triploid plants” refers to plants or transplants derived from plantingtriploid seeds or from micropropagation that have three sets ofchromosomes in the somatic cells, or three times the haploid number.

“Tetraploid plants” are plants or transplants derived from plantingtetraploid seeds or from micropropagation that have four sets ofchromosomes in the somatic cells, or four times the haploid number.

The term “firm flesh” refers to the edible flesh of a watermelon forwhich fruit firmness, as measured using a penetrometer by the methodsdescribed in Example 2, is greater than about 1.5 lbf of pressure butless than or equal to about 2.0 lbf. Botanically, the edible flesh of awatermelon fruit is placental tissue.

The descriptor “ultra firm flesh” refers to the edible flesh of awatermelon with fruit firmness, as measured using a penetrometer by themethods described in Example 2, measuring not less than 3.0 lbf ofpressure, or with higher firmness than fruit produced by standard knowncultivars. Ultra-firm flesh watermelon preferably has fruit firmness ofabout 3.5 lbf.

The term “very firm flesh” refers to the edible flesh of a watermelonwith firmness, as measured using a penetrometer by the methods describedin Example 2, greater than about 2.0 pound force of pressure but lessthan 3.0.

The term “liquid-retaining flesh” refers to edible flesh of a watermelonwhich, once cut, loses less than about four percent of its weight afterthree days storage at 4° centigrade, or retains more liquid, over time,than fruit produced by standard known cultivars. About 95-98% of theweight lost from cut watermelon fruit is estimated to be due to liquidleakage. The majority of the remaining weight loss is from solublesolids, such as sugars and acids. Therefore, liquid loss may beapproximated by measuring the percent weight loss of watermelon fruit,once cut, over time.

A “penetrometer” is a device designed to measure force and is usedherein to measure fruit firmness. It provides a quick, easy and accuratemethod to determine fruit flesh and skin firmness. Applicants gatheredthe data reported herein using a hand-held penetrometer to obtain threeto five pressure readings on mature fruit. Specifically, Applicants usedPenetrometer model FT011 (QA Supplies, Norfolk, Va.) with an 8millimeter, or approximately 5/16 inch, probe.

“Pounds force”, or “lbf”, is the unit read by the penetrometer modelFT011, and is used herein exclusively to indicate readings made usingthe 8 millimeter probe, unless otherwise indicated.

Coloration of the rind in watermelons, also referred to as “rindpattern”, can vary from light green, often termed gray, to medium green,to very dark green, appearing to be almost black. In addition, the rindmay have stripes of various designs which are typical of a variety ortype. Therefore, the terms “tiger stripe”, “mottle stripe”, “dark mottlestripe”, and the like, are used to identify various patterns.

As used herein, “length to width ratio (L/W ratio)” means the ratiosobtained in any of the possible combinations by taking the averagelength divided by the average width on the watermelon fruit. The ratioscan vary from 1:1.2 to 2.2:1.

The term “population” refers to genetically heterogeneous collection ofplants sharing a common parental derivation.

As used herein, the term “variety” or “cultivar” refers to a group ofsimilar plants that, by their genetic pedigrees and performance, can beidentified from other varieties within the same species.

“Backcrossing” refers to the process in which a breeder crosses a plantwith one of its parent lines.

“Recurrent backcrossing” is a breeding strategy designed to recover thegenetic composition of a line by crossing a plant in succession back toone of the parent lines.

The term “soluble solids” refers to the percent of solid material foundin edible fruit. As used herein, soluble solids are measuredquantitatively with a refractometer as percentage brix. Refractometersoften include a sucrose scale, as brix is formally defined as weightpercent sucrose. If the only soluble solid present in an aqueoussolution is sucrose, the sucrose scale should give the actual percentagesucrose. However, if other soluble solids are present, as is almostalways the case, the reading is not equal to the percentage sucrose, butapproximates the overall percentage soluble solids in the sample. Inshort, although brix is technically defined as weight percent sucrose,those of skill in the art recognize that weight percent soluble solids,as obtained with a refractometer, approximates weight percent sucroseand accurately indicates sweetness. Therefore, the higher the percentagesoluble solids, as indicated by brix level, the higher the perceivedsweetness of the fruit.

The U.S. Department of Agriculture has established watermelon fruitquality standards based on brix levels (United States Standards forGrades of Watermelon, U.S. Department of Agriculture (1978)). Accordingto these standards and as used herein, edible parts of the fruit havingnot less than 8 brix are referred to as “good”, while edible parts ofthe fruit having not less than 10 brix are referred to as “very good.”

“Sweetness”, as used herein, may be measured quantitatively, asdescribed above, using a refractometer, or qualitatively, by taste.

A “quantitative trait loci”, or “QTL” is a chromosomal location thatencodes for alleles that affect the expressivity of a continuouslydistributed phenotype.

“Maturity” refers to maturity of fruit development and indicates theoptimal time for harvest. Generally, growers of skill in the art harvestfruit at or substantially near its maximum sweetness and flavorintensity. In watermelon, the maturity comes associated with changes inrind appearance, flesh color and sugar content.

The terms “homozygous” and “homozygosity” are genetic terms. Whenidentical alleles reside at corresponding loci on homologouschromosomes, that locus is called homozygous. Homozygosity typicallyrefers to the degree to which a population is fixed at one or more loci.

A “hybrid” is an offspring of a cross between two genetically unlikeindividuals.

An “inbred” or “inbred line” is a substantially homozygous individual orvariety.

“Introgress” is the process a breeder performs to introduce a new trait,usually from a non-cultivated type, into a cultivated type.

Typical Characteristics of Commercial Watermelon Fruit

Successful watermelon production depends on attention to variouscultural practices. These include soil management, with specialattention to proper fertilization; crop establishment, with appropriatespacing; weed control; the introduction of bees for pollination and, ifproducing fruit from triploid plants, a suitable pollen source forproducing seedless (triploid) watermelon; irrigation; and pestmanagement. Watermelon fruit size and shape, rind color, thickness andtoughness, seed size, color and number, flesh color, texture, sugarcontent and freedom from fruit defects are all important characteristicsto be considered in selection of watermelon varieties. Commercial seedcompanies typically offer the grower an opportunity to observe thesecharacteristics in demonstration plots of their varieties, and someagricultural universities perform cultivar analysis data for the localgrowers (Roberts et al. (2004), Maynard and Sidoti (2003), Schultheisand Thompson (2004) and Leskovar et al. (2004)).

Watermelon crops can be established from seed or from transplants.Transplanting is becoming more common because transplanting can resultin an earlier crop compared with a crop produced from direct seeding.When a grower wants to raise a seedless fruited crop, transplanting ispreferred. Transplanting helps achieve complete plant stands rapidly,especially where higher seed costs, as with triploid seeds, makedirect-seeding risky.

Watermelon is the only economically important cucurbit with pinnatifid(lobed) leaves; all of the other species have whole (nonlobed) leaves.Watermelon growth habit is a trailing vine. The stems are thin, hairy,angular, grooved, and have branched tendrils at each node. The stems arehighly branched and up to 30 feet long (Wehner et al. In: Watermelons:Characteristics, Production and Marketing. Maynard, editor. ASHS Press,Alexandria Va. 2001).

Watermelon breeders are challenged with anticipating changes in growingconditions, new pathogen pressure, and changing consumer preferences.With these projections, a breeder will attempt to create new cultivarsthat will fit the developing needs of growers, shippers, retailers, andconsumers. Thus the breeder is challenged to combine in a singlegenotype as many favorable attributes as possible for good growing,distribution, and eating. One important characteristic for the breederis fruit size. Fruit size is an important consideration because thereare different market requirements for particular groups of shippers andconsumers. The general categories are: icebox (<12 lb), small (12-18lb), medium (18-24 lb), large (24-32 lb), and giant (>32 lb). Fruit sizeis inherited in polygenic fashion, with an estimated 25 genes involved.Fruit is distributed from the grower to the retailer by shippers, whofocus on particular weight categories, such as 18-24 lb for seeded and14-18 lb for seedless. Although historic consumption has been for thesegeneral categories of sizes, there is an increasing trend in themarketplace for a new class of small-fruited watermelon hybrids (withfruit weight between 3-9 lb).

Fruit flesh firmness and liquid retention are other importantcharacteristics. Consumers have varying textural preferences forwatermelon fruit, and flesh firmness is a determinant of texture.Additionally, flesh firmness is a critical parameter that determines howlong cut fruit will last on the retailer's shelf. Liquid retention isalso critical to consumer perception of minimally processed watermelon.Cut fruit shelf life research is usually qualitative, with evaluationson when the fruits become ‘slimy’ (Perkins-Veazie et al. 1998HortScience 33: 605). Quantitative evaluations of cut fruit shelf lifeinclude measuring the flesh firmness directly, using a penetrometer, ormeasuring percent weight loss of cut fruit over time in order toapproximate liquid leakage, as described in Example 4.

Applicants were also able to determine the firmness of various fruitsimply by eating them. Indeed, this was how applicants first determinedthat the watermelons of this invention have ultra firm flesh compared toprior art watermelons. In taste tests, Applicants also determined thatstandard cultivars of the prior art, such as Seminis' diploid Royal Starline, have firm flesh, while the following lines have firm to very firmflesh: Tri-X Brand 626 (Syngenta/Rogers—triploid), Extazy(Hazera—triploid) and Solitaire (Golden Valley—triploid).

Another important fruit characteristic is quality, which includessweetness and attractiveness of fruit and rind color. Wehner et al.((2001) in: Watermelons: Characteristics, production and marketing.Maynard, editor. ASHS Press. Alexandria, Va.) describe thesecharacteristics. Among the most important of these characteristics issweetness, without a bitter taste, which is measured by brix and bytaste. Taste panel data demonstrated a direct correlation of good flavorscores with higher brix levels (Nip et al. (1968) Proc. Amer. Soc. Hort.Sci. 93:547). Brix levels increase as the fruit develops and ripens onthe vine. Thus, immature fruits will have unacceptably low sweetness tothe consumer; if picked too early, the edible tissue will also not haveuniform color. Quantitative recommendations for watermelon fruits havebeen published. While Wehner et al. suggest brix levels between 10% and14% brix, the United States Department of Agriculture has establishedstandards, as described in detail in the “Definitions” section, in whichsweetness of at least 8 brix is good and sweetness of at least 10 brixis very good. Despite some variation in the recommendation and thestandards, there is no dispute that fruit sweetness is a criticalcharacteristic of watermelon fruit.

Characteristics of Watermelon Fruit of the Present Invention

Fruit Firmness

The flesh of watermelon plant fruits of the present invention is firmerand retains liquid better than the fruit flesh of watermelon cultivarsof the prior art. In prior art watermelon fruit, mature edible fleshfrom diploid genotypes are softer than both triploid and tetraploidgenotypes. Fruit firmness variation within a line, irrespective ofploidy level, is insignificant. In general, standard diploid cultivarsproduce fruits with soft to at best firm flesh (i.e., flesh firmness atmaturity from less than 1.0 lbf to about 1.5 lbf). Standard tetraploidlines typically produce fruit with firm flesh or very firm flesh (i.e.,flesh firmness between 1.5 lbf to less than about 3.0 lbf at maturity).Standard triploid hybrids produce seedless fruit with an intermediatelevel of flesh firmness at maturity, ranging from about 1.3 lbf to 2.5lbf. Table 1 shows flesh firmness data from the prior art for commercialhybrids and inbred watermelon lines.

All firmness measurements herein were made using a model FT011penetrometer from QA Supplies in Norfolk, Va. with an 8 millimeter probediameter. Readings were made and are reported in pounds force, a BritishEngineering measurement for pressure, which is abbreviated lbf and isconverted to Newtons according to the following formula: 1 lbf=4.448Newtons. Subject fruits were cut equatorially, midway between theblossom and stem ends of each fruit. Applicants made three to fivereadings per fruit, taking samples from the center of each cut fruit.Reported firmness data is an average of these three to five readings.

TABLE 1 Survey of firmness in typical watermelon cultivars and inbredlines. Average firmness readings are in pound force by methodologydescribed herein. Line Origin Ploidy Firmness Tri-X 313 Syngenta/RogersTriploid 1.4 Millionaire Harris Moran Triploid 1.8 Revolution SunSeedsTriploid 1.7 Majestic Seminis Triploid 1.7 Olympia Seminis Triploid 1.6Omega Seminis Triploid 1.5 PS110-5288-9 Seminis Triploid 2.3 4082Seminis Tetraploid 2.0 4084 Seminis Tetraploid 1.5 4090 SeminisTetraploid 1.6 4133 Seminis Tetraploid 2.2 4134 Seminis Tetraploid 2.44135 Seminis Tetraploid 2.2 4137 Seminis Tetraploid 2.7 4138 SeminisTetraploid 2.2 47602A Seminis Diploid 1.5 4203 Seminis Diploid 1.4Cooperstown Seminis Triploid 1.5 Fenway Seminis Triploid 2.1 SentinelSeminis Diploid 1.4 W-1128 Seminis Diploid 1.4 W-1119 Seminis Diploid1.6 BSI 2532 Seminis Diploid 1.7 BSI 2527 Seminis Diploid 1.3 W-2068Seminis Diploid 1.1 W-2741 Seminis Diploid 1.3 W-1488 Seminis Diploid1.7 BSI 2543 Seminis Diploid 1.2

Compared to prior art watermelon lines, the fruit of the presentinvention both have ultra firm flesh and are sweet. Table 2 displaysflesh firmness and sugar content from watermelon line PI296341, whichwas used as the source of the novel firm flesh fruit of this invention,and hybrid lines created according to the methods described herein.Sweetness measurements were determined quantitatively, using arefractometer (Leica Microsystems Model AR200, Reichert Inc., Depew,N.Y.), according to manufacturer's instructions. One measurement wastaken from each half of an equatorially cut fruit. The data wererecorded as an average.

As indicated by comparing the firmness readings in Table 2 to those inTable 1, the flesh of the watermelon fruit of the present invention isconsiderably more firm than the flesh of the watermelon fruit of theprior art. Specifically, watermelon fruit of the present inventionresist pressure of at least about 3.0 lbf, preferably at least about 3.5lbf, more preferably at least about 4 lbf and most preferably at leastabout 5 lbf.

In addition, as shown in Table 2, watermelon fruit of the presentinvention are sweet. Specifically, watermelon fruit display sweetness ofat least about 6 brix, more preferably at least about 8 brix and mostpreferably at least about 10 brix.

TABLE 2 Firmness and sugar content of inbred and hybrid lines developedfrom the invention described herein and the PI296341 source. Firmnessreadings are in pound force and sugar content is reported as % Brix.Both measurement methods are described herein. Line Origin PloidyFirmness Sugar content PI296341 USDA collection Diploid 13.5 1.6 7132This invention Triploid 4.7 10.2 7133 This invention Triploid 6.2 11.74201 This invention Diploid 8.0 9.7 4203 This invention Diploid 7.8 10.84204 This invention Diploid 6.5 9.7 4207 This invention Diploid 6.5 10Liquid Retention

The fruit of the present invention also retain liquid better than thefruit of the prior art. Example 4 describes a study that demonstratesthis liquid-retaining trait. The study compares liquid leakage rates ofcut fruit from watermelon of this invention and of the prior art whenstored at 4° centigrade. The results of this study are illustrated inFIG. 2. The study measures percent weight loss over time of cut fruit.This measurement approximates liquid loss, as 95-98% of the weight lossis due to liquid leakage. The remaining weight loss is due to leakage ofother components of the fruit, such as soluble solids and acids. Theprimary conclusion from these data is that processed watermelon fruit ofthe present invention lose less liquid over time than processed fruit ofstandard known cultivars.

Watermelon fruit of the present invention lose less than about fourpercent weight after three days storage at 4° centigrade. Preferably,the fruit of the present invention lose less than about three andone-half percent weight after three days storage at 4° centigrade, morepreferably less than about three percent weight, even more preferablyless than about two percent weight, and most preferably less than aboutone and one-half percent weight. Watermelon fruit of the presentinvention also lose less than about five percent weight after a week ofstorage at 4° centigrade. Preferably, the fruit of the present inventionlose less than about four percent weight after a week of storage at 4°centigrade, more preferably less than about three percent weight, evenmore preferably less than about two and one-half percent weight.

In addition to having liquid-retaining flesh, the fruit of the presentinvention are sweet. Specifically, these watermelon fruit displaysweetness at least about 6 brix, more preferably at least about 8 brixand most preferably at least about 10 brix.

Other Traits

Watermelon plants of this invention may be seeded or seedless. Methodsfor obtaining diploid, triploid and tetraploid plants are well known inthe art. Specifically, methods for obtaining diploid and triploidwatermelon plants and seed of the present invention are described indetail below. Tetraploid plants of the present invention may be easilyobtained by those of ordinary skill in the art using known cell biologytechniques and the diploid plants described below.

Using standard crossing techniques, those of skill in the art may obtainwatermelon fruit of the present invention with desirable traits besidesthose described above, as the ultra firm flesh and liquid-retainingflesh traits are dominantly inherited. For example, breeders may easilyobtain watermelons of the present invention that are of a particularsize or have a particular flesh color or rind pattern.

Breeding Techniques—Inbred and Hybrid Lines

Watermelon lines of the present invention were developed in the UnitedStates (Georgia, Florida and California), Mexico and Guatemala beginningin the year 2000. Furthermore, watermelon lines were grown for fieldperformance and evaluation of adaptation in Florida, Georgia andCalifornia beginning in the year 2003. Additionally, diploid andtriploid watermelon hybrids made with lines that produce watermelonshaving ultra firm flesh and/or liquid-retaining flesh at maturity wereevaluated in field conditions in Florida, California and Mexico in 2003and 2004. Specific crosses and firmness and quality evaluations ofresultant fruits are described in detail in the “Examples” section.

For most breeding objectives, commercial breeders work with germplasmthat is often referred to as the ‘cultivated type’. This germplasm iseasier to breed with because it generally performs well when evaluatedfor horticultural performance. The performance advantage the cultivatedtypes provide is sometimes offset by a lack of allelic diversity. Thisis the tradeoff a breeder accepts when working with cultivatedgermplasm—better overall performance, but a lack of allelic diversity.Breeders generally accept this tradeoff because progress is faster whenworking with cultivated material than when breeding with geneticallydiverse sources.

In contrast, when a breeder makes either wide intra-specific crosses, orinter-specific crosses, a converse tradeoff occurs. In these examples, abreeder typically crosses cultivated germplasm with a non-cultivatedtype. In these crosses, the breeder can gain access to novel allelesfrom the non-cultivated type but has to overcome the genetic dragassociated with the donor parent. Because of the difficulty with thisbreeding strategy, this approach often fails because of fertility orfecundity problems. The difficulty with this breeding approach extendsto many crops, and is exemplified with an important disease resistantphenotype that was first described in tomato in 1944 (Smith, Proc. Am.Soc. Hort. Sci. 44:413-416). In this cross, a nematode diseaseresistance was transferred from L. peruvianum (PI128657) into acultivated tomato. Despite intensive breeding, it was not until themid-1970s before breeders could overcome the genetic drag and releasesuccessful lines carrying this trait. Indeed, even today, tomatobreeders deliver this disease resistance gene to a hybrid variety fromonly one parent. This allows the remaining genetic drag to be masked.The inventiveness of succeeding in this breeding approach has beenrecognized by the USPTO (U.S. Pat. Nos. 6,414,226, 6,096,944, 5,866,764,and 6,639,132).

In watermelon, the plant introduction (PI) accessions are typicallylines that produce small fruits with firm white flesh and very poortaste (even bitter). Even though these lines have such poorhorticultural qualities, some watermelon breeders, like some other cropbreeders, attempt to breed with these PI lines because they potentiallycontain novel alleles. To date, the most commonly attempted breedingobjective for use of the PI line series is to introgress new diseaseresistance genes. The process of introgressing novel resistance genesfrom the PI lines into acceptable commercial types is a long and oftenarduous process. This process can be difficult because the trait may bepolygenic, have low heritability, have linkage drag or some combinationof the three.

This breeding project began with a wide cross between cultivatedwatermelons and PI No. 296341, which was obtained from the USDAcollection at the Regional Plant Introduction Station in Griffin, Ga.This accession has been available to watermelon breeders since itsdeposit into the U.S. Plant Introduction system in 1964.

The original intent of the project, however, was not to make watermelonfruit with firm flesh and/or liquid-retaining flesh. Rather, theoriginal intent of the project was to introgress a resistance toFusarium wilt, specifically to Fusarium oxysporum f. sp. niveum race 2,referred to herein as FON race 2. Although no commercial watermelonscurrently contain resistance to FON race 2, the possibility of usingPI296341 as a source of resistance has been known for many years (Netzer(1989) Plant Disease 73:518; Martyn and Netzer (1991) HortScience26:429-432; Wehner et al. ((2001) in: Watermelons: Characteristics,production and marketing. Maynard, editor. ASHS Press. Alexandria, Va.).That there are no watermelon commercial lines for sale with FON race 2resistance introgressed from PI296341, despite these reports as long as15 years ago, underscores the difficulty of introgressing traits fromwide crosses and creating commercially successful inbreds and hybrids.

In addition to being resistant to FON race 2, PI296341 is characterizedby having very small round fruits between 4 and 6 inches in diameter andweighing between 1 and 2.6 pounds. Fruit flesh is white and very firm,with low soluble solids content (Table 2). Organoleptic evaluations ofthese fruits range from no perception of sweetness to bitter. Asdescribed in the “Examples” section below, inbred watermelon plants ofthe present invention may be obtained by crossing a watermelon with theultra firm flesh trait and/or liquid-retaining flesh trait (ultra firmparent) with a non-ultra firm flesh watermelon with other desirablequality characteristics, including sweetness (recurrent parent). Theultra firm parent may be plant introduction accession number 296341.

Those of skill in the art will be able to introgress the ultra firmflesh trait and/or the liquid-retaining trait into the recurrent parentby conducting various recurrent backcrosses, selecting for the (i) ultrafirm flesh and/or liquid-retaining flesh trait and (ii) the sweetnesstrait, and finally self-pollinating selected plants of the recurrentbackcrosses to create inbred watermelon lines with the above traits. Onepossible method for accomplishing such introgression is described in the“Examples” section below.

Applicants generated inbred line 3347, which generates sweet ultra firmfruit according to the present invention, using the methods describedabove and in the “Examples” section. See, especially, Example 5. Inbredline 3347 has been deposited with NCIMB and accorded Accession No. NCIMB41230. Details of the deposit follow the “Examples” section.

Using known methods, breeders may obtain diploid, triploid andtetraploid inbred lines of watermelon having fruit with the (i) ultrafirm flesh and/or liquid-retaining flesh trait and (ii) sweetness trait.

In addition, because the ultra firm flesh and liquid-retaining traits ofthe present invention are dominantly inherited, breeders may obtainhybrids using the watermelons of this invention. Hybrids may be eitherdiploid or triploid. Specifically, breeders crossed inbred watermelonplants with the above desired flesh traits and sweetness traits toeither diploid or tetraploid non-ultra firm flesh cultivars to create,respectively, diploid and triploid watermelon plants with fruit havingthe ultra firm flesh and/or liquid-retaining flesh trait and sweetnesstrait. The non-ultra firm flesh parent used in creating a hybrid mayalso be used to obtain sweet ultra firm flesh and/or liquid-retainingflesh watermelon with other desirable traits, such as a particular sizeand/or color.

Those skilled in the art recognize that there are several breedingmethods used for the introgression of new traits into commercialgermplasm, including mass selection, pedigree selection, recurrentselection and backcrossing. By way of example, and by no means limiting,the introgression of ultra firm flesh watermelon fruit at maturity, withhigh brix levels is described below.

EXAMPLES Example 1 Generation of F1 Lines and Backcrosses

In the summer of 2000, four first filial (F1) generation lines werecreated by crossing 4 Seminis inbred lines as females to PI296341. Thefour diploid inbred lines used were W-2388, W-1128, W-1119 and W-1488.Line W-2388 is elongated in shape with a length to width (L/W) ratio of1.8 to 2.2:1. The rind color and pattern is of medium green backgroundwith wide darker stripes. This shape and rind pattern phenotype is knownto those skilled in the art as an “elongated dark mottle stripe”watermelon fruit. The fruit shape of Line W-1128 is round oval with L/Wratio of 1.0-1.2:1 and rind color is of light to medium green backgroundand narrow darker green stripes. This phenotype is known to thoseskilled in the art as “round-oval with narrow (or tiger) stripes”watermelon fruit. Fruit shape of Line W-1119 is oval to high round withL/W ratio of 1.1-1.3:1. Rind color is medium green background with widedarker green stripes. This phenotype is known to those skilled in theart as “round-oval dark mottle stripe” watermelon fruit. Fruit of LineW-1488 is of round shape with L/W ratio of 1.0 to 1.1:1. Rind color islight green with some faint mottle/net pattern in the background. Thisphenotype is known to those skilled in the art as “round gray (or lightgreen)” watermelon fruit. These four lines provide an array ofphenotypic diversity amongst the cultivated types.

In the fall of 2000, the respective F1s were used as females tobackcross to the above four inbreds, creating the backcross 1 (BC1)generation.

The BC1 generation plants were grown in the spring of 2001, andselections were made based on overall vigor. It was difficult to takethe alleles from the PI line into the cultivated types because many ofthe BC1 and even BC2 plants died. Variation in vine vigor was observedthat was associated with survivability. Vine vigor was assumed to beassociated with general vigor, and perhaps with pathogen resistance.

The respective BC1 lines, derived from the original four inbreds werecrossed as females as follows:

-   -   1. [[W-1128×PI296341]F1×W-1128](this is the W-1128 BC1)×W−1128    -   2. [[W-1119×PI296341]F1×W-1119](this is the W-1119 BC1)×W−1119    -   3. [[W-1488×PI296341]F1×W-1488](this is the W-1488 BC1)×W−1488    -   4. [[W-2388×PI296341]F1×W-2388](this is the W-2388 BC1)×W−2068    -   5. [[W-2388×PI296341]F1×W-2388](this is the W-2388 BC1)×BSI−2543    -   6. [[W-2388×PI296341]F1×W-2388](this is the W-2388 BC1)×BSI−2527

In these six crosses, the first three were recurrent parent backcrosses.Cross number four was to line W-2068, which is very similar to originalparent W-2388. Crosses five and six were to new inbreds. The recurrentbackcross program aims to add one or more new traits from the donorparent (in this case, PI296431), while retaining the phenotype of therecurrent parent. However, watermelon breeding is a dynamic process, soit is not uncommon to change the recurrent parent as newer inbred linesare being developed concurrently. Crosses four through six, therefore,were not technically creating the BC2 generation. For clarity indescribing the generations, these crosses will be referred to as theBC2* generation. The BC2 and BC2* generation were grown in the summer of2001. As with the BC1 generation, selection for vine vigor was made.Females thus selected were used to create the BC3 and BC3* generation.

-   -   1. W-1128 BC2×W-1128=BC3    -   2. W-1119 BC2×W-2741=BC3*    -   3. W-1488 BC2×W-1488=BC3    -   4. W-2068 BC2*×W-2068=BC3*    -   5. BSI-2543 BC2*×BSI-2543=BC3*    -   6. BSI-2527 BC2*×BSI-2527=BC3*        In the fall of 2001, the BC3 and BC3* lines were grown, and        selection was again applied for vine vigor. Selected plants were        then crossed to create the BC4 and BC4* generations,        respectively.    -   7. W-1128 BC3×W-1128=BC4    -   8. W-2741 BC3*×W-2741=BC4*    -   9. W-1488 BC3×W-1488=BC4    -   10. W-2068 BC3*×W-2068=BC4*    -   11. BSI-2543 BC3*×BSI-2543=BC4*    -   12. BSI-2527 BC3*×BSI-2527=BC4*

In addition to selecting for vine vigor, examination of BC3 and BC3*fruit, which contained the BC4 and BC4* generation seed, produced anunexpected finding. Although the BC3 generation still performs poorlywhen evaluated by current horticultural characteristics, the fruits wereexamined for quality characteristics. Although most fruit had poorquality, breeder observations as to a small number of fruit included thefollowing: “good fruit color, sweet taste and ultra firm flesh—like anapple.” The unexpected finding was that both ultra firm flesh and sweettasting flesh could be created. The possibility of creating a sweettasting flesh, combined with ultra firm flesh for the cut fruit segmentof the marketplace resulted in a bifurcation of breeding objectives.Applicants initiated a new project with the goal of creating ultra firmflesh watermelon fruits with sweet taste.

Example 2 Self-Pollinations of Plants Bearing Ultra Firm Flesh Fruit andEarly Flesh Firmness Data

In the spring of 2002, the BC4/BC4* generation was grown and evaluatedqualitatively for sweet taste, fruit flesh firmness, and horticulturalcharacteristics. Based on these evaluation criteria, plants wereselected to create the next generation. Instead of creating anotherbackcross generation, however, each selection from the lines beingdeveloped in parallel was self pollinated. The crossing produced theBC4S1/BC4*S1 generation.

In the summer of 2002, the BC4S1/BC4*S1 generation was grown andevaluated qualitatively for sweet taste, fruit flesh firmness, andhorticultural characteristics. Based on these evaluation criteria,plants were selected to create the next generation. Self pollination ofthe selected plants created the BC4S2/BC4*S2 generation.

In the fall of 2002, the BC4S2/BC4S*2 generation was grown and evaluatedqualitatively for sweet taste, fruit flesh firmness, and horticulturalcharacteristics. Based on these evaluation criteria, plants wereselected to create the next generation. Self pollination of the selectedplants created the BC4S3/BC4S*3 generation.

In the spring of 2003 the BC4S3/BC4S*3 generation was grown andevaluated qualitatively for sweet taste, fruit flesh firmness, andhorticultural characteristics. Based on these evaluation criteria,plants were selected to create the next generation. Self pollination ofthe selected plants created the BC4S4/BC4S*4 generation.

For the BC4S3 fruit, both qualitative and quantitative data wereobtained for flesh firmness. Specifically, ninety three fruits fromindividual BC4S3 plants were evaluated for firmness with a penetrometer(model FT011 with an 8 millimeter probe, QA Supplies, Norfolk, Va.). TheFT011 penetrometer has a gauge that reads PF, which is an improperabbreviation for pound force. Pound force is a British Engineeringmeasurement scale for pressure, and is properly abbreviated lbf. Theconversion from the British measurement system to the InternationalSystem of Units (SI) is 1 lbf=4.448 newtons. For all flesh firmnessmeasurements using a penetrometer, mature fruits were detached from theplant and cut in an equatorial direction. For orientation, fruits have astem end and a blossom end. Equatorial slicing means that the fruits arehalved such that each half has the blossom end or stem end the farthestdistance from the cut site. Samples were taken from the center of thecut fruit. For diploid fruits, sampling occurred inside the seeded ring.Although triploid fruits have few to no seeds, sampling occurred withinthe same core area of the split fruit. Each half was sampled with thepenetrometer, with a total of three to five readings per fruit. Firmnessdata are reported as an average of the three to five readings.

Even after several generations attempting to fix the firm flesh genotypecombined with acceptable horticultural characteristics, includingsweetness, FIG. 1 shows that significant fruit flesh firmness variationstill existed in these samples. Although the data in FIG. 1 indicatesignificant variation, it was clear that improvements to fruit firmnesshad been made. The arrow shows the average firmness rating of therecurrent parents. Even at this early generation in product development,approximately 43% of the fruits have firmness measurements of not lessthan 4 lbf.

Some phenotypes are determined by the genotype at one locus. Thesesimple traits, like those studied by Mendel, fall in discontinuouscategories such as green or yellow seeds. Most variation observed innature, however, is continuous, like yield in field corn, or human bloodpressure. FIG. 1 shows a continuous-type pattern of firm fleshvariation, similar to a normal distribution. Unlike simply inheritedtraits, continuous variation can be the result of polygenic inheritance.Loci that affect continuous variation are referred to as quantitativetrait loci, or QTLs. Variation in the phenotype of a quantitative traitis the result of the allelic composition at the QTLs and anenvironmental effect. Applicants identified several potential causes forthe variation: (1) the fruit firmness trait may be controlled by severalto many QTLs; (2) the fruit firmness trait may be caused by one or a fewgenes, but have a low heritability; and (3) the trait may be bothpolygenic and have low heritability. Those skilled in the art recognizethat the marketplace requires product uniformity. Thus, the utility ofthe invention is higher for those traits with high heritability that arenot greatly affected by the environment. The heritability of a trait isthe proportion of the phenotypic variation attributed to the geneticvariance. This ratio varies between 0 and 1. Thus, a trait withheritability near 1.0 is not greatly affected by the environment.Because the fruit firmness variation shown in FIG. 1 did not explain thecause of the variation, further experiments were conducted, as describedin the examples below, to determine the cause of the variation.

Example 3 Generation of Diploid Hybrids with Ultra Firm Flesh Trait

In the fall of 2002, in addition to the self pollinations, crosses withselected BC4S2/BC4S*2 generation plants were made to other commercialinbreds that do not contain the ultra firm flesh phenotype. Thesecrosses were made to test to what extent the ultra firm flesh traitwould be dominantly inherited in a hybrid combination. Those skilled inthe art will recognize the importance of establishing how well traitsdeveloped in inbred lines function in a hybrid combination.

In the spring of 2003, these test hybrids were evaluated in Florida andCalifornia. Although many hybrid combinations were tested in thesetrials, most of these data are not shown. Instead, data from four topperforming hybrids across two trialing locations are shown in Tables 3and 4. Hybrids were evaluated by a number of criteria, including therind color pattern. For these hybrids, all had a mottled stripe pattern,designated MS. Also evaluated were fruit length and width, rindthickness, flesh color, firmness and sweetness levels.

When determining sweetness levels quantitatively, Applicants used arefractometer to measure brix levels. Specifically, brix levels weremeasured with a digital, hand-held refractometer (Leica Microsystemsmodel AR200, Reichert Inc., Depew, N.Y.) according to manufacturer'sinstructions. Brix levels were determined after the penetrometerfirmness readings, by squeezing a sampled fruit until drops of liquidfell into the well of the refractometer. One brix measurement was takenfrom each half of a cut fruit, and the data were recorded as an average.

Tables 3 and 4 show that the test hybrids do exhibit small variationbetween the test sites. Taken together, however, the data show thatthese top performing hybrid combinations performed uniformly in the twolocations. In particular, these hybrids consistently had ultra firmflesh, as measured by pound force of pressure and very good solublesolids, as measured by percentage brix.

Fruit flesh firmness data across the two locations provided insight intothe genetics of the trait, answering questions as to heritability posedby the data shown in FIG. 1. First, these data show that the ultra firmflesh trait can be delivered into an F1 hybrid from a single parent. Inother words, genetic loci selected in the method described herein affectfruit firmness in a dominant manner. This is a critical fact for thedesign of breeding strategies. Moreover, consistency in the firmnessmeasurements across several hybrids in the two locations show that theultra firm flesh alleles selected in the method described herein have ahigh heritability. Those skilled in the art recognize the importance ofcreating commercial lines with highly heritable horticultural traits.Specifically, such cultivars will allow growers to produce a crop withuniform market specifications.

TABLE 3 Test hybrid evaluations: Florida, Spring 2003 Rind Firm- LengthWidth Thickness Flesh ness Sweetness Hybrid Rind (cm) (cm) (cm) Color(lbf) (Brix) 4201 MS 23 19.5 1.5 Red 8.0 9.7 4203 MS 25.5 21.5 1.5 Red7.5 11.3 4204 MS 23 19 1.0 Red 6.0 9.3 4207 MS 25 20 2.0 Red 7.0 9.6

TABLE 4 Test hybrid evaluations: California, Spring 2003 Rind Firm-Length Width Thickness Flesh ness Sweetness Hybrid Rind (cm) (cm) (cm)Color (lbf) (Brix) 4201 MS 22.5 17 1.5 Red 8.0 9.7 4203 MS 23 17 1.5 Red8.0 10.3 4204 MS 25 18 2.0 Red 7.0 10.0 4207 MS 25 17 1.5 Red 6.0 10.3

Example 4 Evaluation of Liquid-Retaining Flesh Characteristics of UltraFirm Flesh Hybrids

As described herein, studies agree that minimally processed productshave a short shelf life of 2 to 3 days maximum (Perkins-Veazie et al.(1998) Hortscience 33:605; Wehner et al. in: Watermelons:Characteristics, Production and Marketing. Maynard, editor. ASHS Press,Alexandria Va. 2001). Although the maximum shelf life of cut watermelonfruits is only a few days, product quality begins to deteriorate rapidlyafter being processed. In cut products presented in plastic foodcontainers, the consumer can see this rapid deterioration because liquidwill leak out of the cut products and accumulate in the bottom of thecontainer.

Mature fruits from the 2003 California hybrid trial (Example 3, Table 4)were evaluated for leakage using a liquid retention test as describedherein (see FIG. 2). This test was performed at 4° centigrade. Fruitsfrom test hybrids 4201, 4204 and 4207 were tested along with standarddiploid and triploid hybrid controls. Test hybrids had the ultra firmflesh trait, with firmness readings of 8.0 lbf, 7.0 lbf and 6.0 lbf,respectively (Table 4). Controls had flesh firmness readings of <2.0 lbfand <2.5 lbf, respectively. To measure liquid loss, the edible portionof the fruits were cut into approximately 1″ cubes and weighed. Theapproximate 1 inch cube size was chosen because this best approximatesthe processed product size found in retail outlets. Over a 16 dayperiod, samples were re-weighed, and the liquid loss was estimated bycalculating the percent weight loss.

FIG. 2 graphically displays the percent weight loss of these samplesover a 16 day period. Multiple samples per line were tested; thetriangles, circle and squares represent the mean values at each timepoint, and the sample standard deviations are shown as bars. Data inFIG. 2 show large differences in weight losses between the controlshaving softer fruit flesh and the fruits with the ultra firm fleshtrait. The difference between the controls and the test hybrids with theultra firm flesh phenotype was apparent by the first time point, whichwas approximately 6½ hours after the samples were cut. Therefore,although cut product from standard cultivars may have a shelf life of upto 2 to 3 days, product deterioration begins almost immediately afterthey are cut. These data show that the ultra firm flesh lines developedusing the method described herein will resist the rapid liquid leakagenow common in cut watermelon fruits. Because these ultra firm fleshfruits will retain liquid once cut, they will last longer in theminimally processed watermelon market.

Example 5 Final Self Pollinations and Creation and Evaluation ofTriploid Hybrids

In the summer of 2003, the BC4S4/BC4S*4 generation, the generation ofwhich is described above in Example 2, was grown and evaluatedqualitatively for sweet taste, fruit flesh firmness, and horticulturalcharacteristics. Based on these evaluation criteria, plants wereselected to create the next generation. Self pollination created theBC4S5/BC4S*5 generation.

In the fall of 2003, the BC4S5/BC4S*5 generation was grown and evaluatedqualitatively for sweet taste, fruit flesh firmness, and horticulturalcharacteristics. Based on these evaluation criteria, plants wereselected to create the next generation. Self pollination created theBC4S6/BC4S*6 generation.

In addition, quantitative firmness data were collected from the BC4S5generation for lines that were qualitatively sweet. Specifically, twentysix lines were tested, and results are shown below in Table 5. Fourteenof these lines had a single fruit tested, and the remaining 12 lines had2 or 3 fruits tested per line. The range of firmness amongst the twentysix lines ranged from a low of 4.0 lbf to a high of 8.0 lbf. For thelines that had multiple samples, 11 of the 12 lines showed no differencein the penetrometer measurements. One line did show a penetrometermeasurement difference of 1 lbf. These data provide further insight asto questions raised by FIG. 1, which showed variation in the ultra firmflesh trait in the BC4S3 generation. In particular, it was unclear inthe BC4S3 generation whether the ultra firm flesh trait displayed a lowor high heritability. That many lines developed in parallel gaveelevated, but different fruit firmness readings suggested that the ultrafirm flesh is polygenic in nature. The very low intra-line variationshown in Table 5, together with the test hybrid data shown in Tables 3and 4 demonstrate that the ultra firm flesh trait has a highheritability. Those skilled in the art recognize the importance ofcreating commercial lines with highly heritable horticultural traitsbecause such cultivars allow growers to produce a crop with uniformmarket specifications. Table 5 shows the inbred line evaluations fromthe BC4S5/BC4S*5 generation.

Line-replication no. Firmness 3333-1 7.0 lbf 3333-2 8.0 lbf 3334-1 5.0lbf 3334-2 5.0 lbf 3335-1 8.0 lbf 3335-2 8.0 lbf 3336-1 5.0 lbf 3336-25.0 lbf 3337-1 5.0 lbf 3339-1 4.0 lbf 3340-1 4.5 lbf 3340-2 4.5 lbf3340-3 4.5 lbf 3341-1 5.0 lbf 3346-1 5.0 lbf 3346-2 5.0 lbf 3347-1 6.0lbf 3347-2 6.0 lbf 3347-3 6.0 lbf 3348-1 6.0 lbf 3348-2 6.0 lbf 3348-36.0 lbf 3349-1 5.0 lbf 3350-1 5.0 lbf 3350-2 5.0 lbf 3350-3 5.0 lbf3352-1 5.5 lbf 3353-1 6.0 lbf 3355-1 8.0 lbf 3355-2 8.0 lbf 3357-1 5.0lbf 3357-2 5.0 lbf 3358-1 5.0 lbf 3358-2 5.0 lbf 3359-1 6.0 lbf 3378-17.0 lbf 3380-1 7.0 lbf 3384-1 7.0 lbf 3386-1 7.0 lbf 3387-1 8.0 lbf3388-1 7.0 lbf 3390-1 6.0 lbf 3390-2 6.0 lbf 3392-1 8.0 lbf 3392-2 8.0lbf 3394-1 5.5 lbf 3394-2 5.5 lbf 3396-1 7.0 lbf 3396-2 7.0 lbf 3397-17.0 lbf 3397-2 7.0 lbf 3398-1 7.5 lbf 3399-1 7.5 lbf 3399-2 7.5 lbf3400-1 7.5 lbf 3401-1 7.5 lbf 3401-2 7.5 lbf 1577-1 8.0 lbf 1577-2 8.0lbf 1577-3 8.0 lbf 1577-4 8.0 lbf 1577-5 8.0 lbf 1577-6 8.0 lbf 1577-78.0 lbf

In addition to the self pollinations described above, crosses withselected BC4S4/BC4S*4 generation plants were made to other commercialtetraploid inbreds that do not contain the ultra firmness phenotype.These tetraploid×diploid crosses were made to test to what extent theultra firm flesh trait would be dominantly inherited in a triploidhybrid combination. As shown in Table 6 below, the ultra firm fleshtrait was inherited by the triploid seedless fruit.

TABLE 6 Mature fruit flesh firmness and sweetness scores. Firmness wasmeasured as described herein with a penetrometer. Length Width RindFlesh Firmness TSS Hybrid Rind (cm) (cm) Thickness (cm) Color (lbf)(Brix) SVR8034-7131 TS 28 24 1.2 Red 5.0 10.2 SVR8034-7132 MS 26 25 1.0Red 4.0 9.7 SVR8034-7133 MS 28 26 1.0 Red 5.0 10.5 SVR8034-7134 MS 26 241.1 Red 4.5 10.0

All references cited herein are hereby expressly incorporated herein byreference.

DEPOSIT INFORMATION

A deposit of the Seminis Vegetable Seeds proprietary inbred and hybridwatermelon line 3347 disclosed above and recited in the appended claimshas been made with NCIMB Ltd, 23 St. Machar Drive, Aberdeen AB24 3RY.The date of the deposit was 1 Jul. 2004. The deposit of 2500 seeds forthis variety were taken from the same deposit maintained by SeminisVegetable Seeds since prior to the filing date of this application. Uponissuance of a patent, all restrictions upon the deposit will be removed,and the deposit is intended to meet all of the requirements of 37 C.F.R.§1.801-1.809. The NCIMB accession numbers for inbred line 3347 wasdeposited as Accession No. NCIMB 41230.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

What is claimed is:
 1. A watermelon plant, or a part thereof, thatproduces a mature fruit having soluble solids of at least 6 brix, andsaid fruit having flesh that resists pressure of at least 3.5 poundsforce (lbf), wherein watermelon line PI296341 or 3347 is the source ofsaid fruit trait having flesh that resists pressure of at least 3.5pounds force (lbf), and wherein a representative sample of seed of saidline 3347 has been deposited with the NCIMB under NCIMB Accession No.41230.
 2. The watermelon plant, or a part thereof, of claim 1, whereinsaid flesh of said mature fruit resists pressure of at least 4.0 lbf. 3.The mature fruit of claim 2, wherein said flesh of said mature fruit hassoluble solids of at least 8 brix, at least 10 brix, or at least 11.5brix.
 4. The watermelon plant, or a part thereof, of claim 1, whereinsaid mature fruit weighs at least 1.5 kg, at least 3.0 kg, at least 4.5kg, or at least 6.0 kg.
 5. The watermelon plant, or a part thereof, ofclaim 1, wherein said mature fruit has red flesh, yellow flesh, ororange flesh.
 6. The watermelon plant, or a part thereof, of claim 1,wherein the plant is diploid, tetraploid, or triploid.
 7. A watermelonfruit harvested from the plant according to claim
 1. 8. Watermelon fleshderived from the watermelon fruit of claim
 7. 9. The watermelon plant,or a part thereof, of claim 1, wherein said plant has a diploid maleparent.
 10. The watermelon plant, or a part thereof, of claim 1, whereinsaid flesh of said mature fruit is liquid-retaining flesh that when cutloses less than three and one-half percent weight after three daysstorage at 4° centigrade, loses less than three percent weight afterthree days storage at 4° centigrade, loses less than two percent weightafter three days storage at 4° centigrade, loses less than one andone-half percent weight after three days storage at 4° centigrade, losesless than five percent weight after a week of storage at 4° centigrade,loses less than four percent weight after a week of storage at 4°centigrade, loses less than three percent weight after a week of storageat 4° centigrade, or loses less than two and one-half percent weightafter a week of storage at 4° centigrade.
 11. A seed that produces awatermelon plant, or a part thereof, of claim
 1. 12. A watermelon plant,or a part thereof, that produces a mature fruit having liquid-retainingflesh, that when cut, loses less than three and one-half percent weightafter three days storage at 4° centigrade, loses less than three percentweight after three days storage at 4° centigrade, loses less than twopercent weight after three days storage at 4° centigrade, loses lessthan one and one-half percent weight after three days storage at 4°centigrade, loses less than five percent weight after a week of storageat 4° centigrade, loses less than four percent weight after a week ofstorage at 4° centigrade, loses less than three percent weight after aweek of storage at 4° centigrade, or loses less than two and one-halfpercent weight after a week of storage at 4° centigrade, whereinwatermelon line PI296341 or 3347 is the source of said liquid-retainingflesh trait, and wherein a representative sample of seed of said line3347 has been deposited with the NCIMB under NCIMB Accession No. 41230.13. The fruit of claim 12, wherein said flesh of said mature fruitresists pressure of at least 3.5 lbf, at least 4.0 lbf, at least 5.0lbf, at least 6.0 lbf, or at least 8.0 lbf.
 14. The watermelon plant, ora part thereof, of claim 12, wherein said flesh of said mature fruit hassoluble solids of at least 6 brix, at least 8 brix, at least 10 brix, orat least 11.5 brix.
 15. The watermelon plant, or a part thereof, ofclaim 12, wherein said mature fruit weighs at least 1.5 kg, at least 3.0kg, at least 4.5 kg, or at least 6.0 kg.
 16. A seed that produces awatermelon plant, or a part thereof, of claim
 15. 17. A hybridwatermelon plant, or a part thereof, that produces a mature fruit havingsoluble solids of at least 6 brix and said fruit having flesh thatresists pressure of at least 3.5 pounds force (lbf), wherein watermelonline PI296341 or 3347 is the source of said fruit having flesh thatresists pressure of at least 3.5 pounds force (lbf), and wherein arepresentative sample of seed of said line 3347 has been deposited withthe NCIMB under NCIMB Accession No.
 41230. 18. A fruit of the watermelonplant, or a part thereof, of claim 17, wherein said flesh of said maturefruit resists pressure of at least 4.0 lbf, at least 5.0 lbf, at least6.0 lbf, or at least 8.0 lbf.
 19. The hybrid watermelon plant, or a partthereof, of claim 17, wherein said watermelon plant, or a part thereof,produces said mature fruit having liquid-retaining flesh that when cutloses less than three and one-half percent weight after three daysstorage at 4° centigrade, loses less than three percent weight afterthree days storage at 4° centigrade, loses less than two percent weightafter three days storage at 4° centigrade, loses less than one andone-half percent weight after three days storage at 4° centigrade, losesless than five percent weight after a week of storage at 4° centigrade,loses less than four percent weight after a week of storage at 4°centigrade, loses less than three percent weight after a week of storageat 4° centigrade, or loses less than two and one-half percent weightafter a week of storage at 4° centigrade.
 20. A fruit of the watermelonplant, or a part thereof, of claim
 19. 21. The hybrid watermelon plant,or a part thereof, of claim 18, wherein said mature fruit has solublesolids of at least 8 brix, at least 10 brix, or at least 11.5 brix, andliquid-retaining flesh.
 22. A fruit of the watermelon plant, or a partthereof, of claim
 21. 23. A seed that produces a hybrid watermelonplant, or a part thereof, of claim
 17. 24. A hybrid watermelon plant, ora part thereof, that produces a mature fruit having liquid-retainingflesh that when cut loses less than three and one-half percent weightafter three days storage at 4° centigrade, loses less than three percentweight after three days storage at 4° centigrade, loses less than twopercent weight after three days storage at 4° centigrade, loses lessthan one and one-half percent weight after three days storage at 4°centigrade, loses less than five percent weight after a week of storageat 4° centigrade, loses less than four percent weight after a week ofstorage at 4° centigrade, loses less than three percent weight after aweek of storage at 4° centigrade, or loses less than two and one-halfpercent weight after a week of storage at 4° centigrade whereinwatermelon line PI296341 or 3347 is the source of said liquid-retainingflesh trait, and wherein a representative sample of seed of said line3347 has been deposited with the NCIMB under NCIMB Accession No. 41230.25. A fruit of the watermelon plant, or a part thereof, of claim 24,wherein said flesh of said mature fruit resists pressure of at least 3.5pounds force (lbf), at least 4.0 lbf, at least 5.0 lbf, at least 6.0lbf, or at least 8.0 lbf.
 26. The watermelon plant, or a part thereof,of claim 24, wherein said flesh of said mature fruit has soluble solidsof at least 6 brix, at least 8 brix, at least 10 brix, or at least 11.5brix.
 27. The watermelon plant, or a part thereof, of claim 24, or apart thereof, wherein said plant is diploid or triploid.
 28. A fruit ofthe watermelon plant, or a part thereof, of claim
 27. 29. Watermelonflesh derived from the watermelon fruit of claim
 27. 30. A seed thatproduces a hybrid watermelon plant, or a part thereof, of claim
 24. 31.The watermelon plant, or a part thereof, of claim 1, wherein said fleshof said mature fruit resists pressure of at least 5.0 lbf.
 32. Thewatermelon plant, or a part thereof, of claim 1, wherein said flesh ofsaid mature fruit resists pressure of at least 6.0 lbf.
 33. Thewatermelon plant, or a part thereof, of claim 1, wherein said flesh ofsaid mature fruit resists pressure of at least 8.0 lbf.
 34. Thewatermelon plant, or a part thereof, of claim 1, wherein said part ofthereof is a seed.